FI92326C - LHRH nonapeptide and decapeptide analogs useful as LHRH antagonists - Google Patents

Abstract

Peptides of formula (I) and pharmaceutically acceptable salts are new: A-B-C-Ser-D-E-F-G-Pro-J (I) where A= aminoacyl from D- or L- isomers of: N-Ac-D,L- delta (3,4)-Pro, N-Ac-D,L-Pro, N-Ac-D,L-Phe, N-Ac-D,L- Pcl-Phe, N-Ac-D,L-PF-Phe, N-Ac-3-(1-naphthyl)-D,L-Ala, N-Ac-3-(2-naphthyl)-D,L-Ala and N-Ac-3-(2,4,6-trimethylphenyl)-D,L-Ala; B = D-Phe, D-Pcl-Phe, D-PF-Phe, D-PNO2-Phe, 2,2-diphenylglycyl, D-alpha-methyl-P-cl-Phe or 3-(2-naphthyl)-D-Ala; C= D-Trp, D-Phe, 3-(3-pyridyl)-D-Ala or 3-(2-naphthyl)-D-Ala; D= L-Phe, L-Tyr, 3-(3-pyridyl)-Ala, Arg or G; E= 3-(2-naphthyl)-D-Ala, 3-(3-pyridyl)-D-Ala, D-Tyr, D-Trp, D-nicotinyl-Lys, pyridylacetyl-Lys, D-Glu(AA) or G; F= L-Leu, L-Nle, L-Phe, L-Trp or 3-(2-naphthyl)-L-Ala; G = gp. of formula (IIa), (IIb) or (IIc); n = 1-5; R1 = 1-6C alkyl or fluoroalkyl; R2 = H or R1; or R1-NH-C=NR2 = ring of formula (IIIa), (IIIb) or (IIIc): m = 1-4; A = H or 1-6C alkyl; X = A or halogen; R3 = H, 1-6C alkyl, phenyl or phenyl-lower alkyl; J = D-Ala-NH2, D-Leu-NH2, Gly-NH2 or -NHR4; R4 = lower alkyl or -NHCONH2; D-On(AA) = anisole adduct to D-Glu to form a P-methoxy-phenylketone (at the carboxyl termius of the glutamic acid side chain), i.e. Glu (pMeO-Ph).

Description

5>. 3 2 6 LHRH nonapeptide and decapeptide analogues useful as LHRH antagonists

Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are released from the anterior lobe of the brain under the control of luteinizing hormone releasing hormone (LHRH, LH / FSH-RH, GnRH) produced in the hypothalamic region. LH and FSH affect gonads by accelerating the synthesis of steroid hormones and the maturation of gametes. The single release of LHRH and thus the release of LH and FSH accompany the reproductive cycle in domestic and human cilia.

LHRH also affects the placenta and through it epa-directly to the gonads by inducing the synthesis and release of choriogonadotropin 15 (OG).

LHRH antagonists are useful in fertilization. Such antagonists inhibit ovulation in females and spermatogenesis in males. These effects are closely related to a decrease in the level of gonad-derived steroid sex hormones from normal circulatory levels, which causes a decrease in accessory organ mass in males and females. In domestic animals, this effect prevents sexual rotation and behavior (promoting weight gain ..25 during the grazing phase), causes miscarriages of pregnant women and usually results in chemical sterilization.

The natural releasing hormone LHRH is a decapeptide composed of naturally occurring amino acids 30 (with glycine, which is an asymmetric-centered amino acid; except for the L-configuration). Its amino acid sequence is as follows: (pyro) Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 4 35 123456789 10 2? V7 "6 Compounds analogous to this natural substance are often shown in abbreviated form by demonstrating the nature of the substitute for a particular amino acid whose position is indicated by a superscript, followed by “LHRH.” Many analogs of LHRH have been studied, and a very large majority of them have insufficient biological activity to be clinically useful. however, the modifications have the effect of enhancing agonistic biological activity A significant increase in agonist activity is achieved by replacing Gly with an D-amino acid residue at the 6-position acid residue.

In addition to agonists, analogs have been prepared which are competitive antagonists of LHRH and all of which require the removal or replacement of the histidyl group in the 2-position Zw · Vale et al., Science 176 (1972) 933 /. In general, a D-amino acid residue appears to produce the best activity at 20 ZR.W.A when placed at that position in the chain. Rees et al., J. Med. Chem. 17 (1974) 10167-

In addition, further modification at the 6-position (which, without 2-position conversion, results in the aforementioned agonist activity) increases the antagonist activity iC of the 2-modified analogs. W. Beattie et al., J. Med.

·. 25 Chem. 18 (1975) 1247; J. Rivier et al., Peptides 1976, Editions de l'Universite de Bruxeller, Belgium, 1976, p. 4277.

On the basis of these two main variants, which lead to more potent LHRH antagonists, the antagonistic activity has been further increased by converting the 1-, 3-, 5- and / or 10-position 7 jo of the already 2,6-modified peptide. H. Coy et al., Peptides 1976, Editions de l'Universite de Bruxelles, Belgium, 1976, s 462; J. E. Rivier et al., Life Sci. 23 (1978) 869; A. S. Dutta et al., 35 Biochem. Biophys. Res. Commun. 85 (1979) 7097 · 0n MySs 1

II

· 3 i? 2 3 2 6 shown that N-acylation of an amino acid residue at position 1 has the advantage / K. Channabasavaia et al., Bio-chem. Biophys. Res. Commun. 81 (1978) 382; D. H. Coy et al., Peptides - Structure and Biological Function, Pier-5 ce Chemical Co., 1979, p. 775 /. In addition, the article /D.H. Coy, Endocrinology 110 (1982) 1445/1 which deals with a highly potent antagonist containing the D-Arg substitution, (N-Ac-D-pCl-Phe, D-pCl-Phe, D-Trp3, D- Arg6, D-Ala10) LHRH.

β 10 Yes, although DHR-containing analogs of LHRH containing the D-Arg substitution have been found to be effective anti-ovulatory agents, they were also potent cells. Cell degranulants / Schmidt et al.,

Contraception 29 (1984) 283 / and caused eczema in 1 2 15 vivo. Thus, for example (N-Ac-D-Nal (2), D-pCl-Phe, -¾ β D-Trp, D-Arg), the ED, -g of LHRH is 0.2 μg / ml, which becomes for the release of amine from rat mast cells in vitro.

This side reaction is clinically important due to the potentially life-threatening nature of the following anaphylactic-type reaction.

It is known in the art that molecules containing positive charge / positive charges, especially multiple positive charges combined with hydrophobicity, are potent mast cell degranulators' 1 25 / Foreman and Jordan, Agents and Action 13 (1983) 1057 · The first attempt to circumvent this the problem with analogs containing two Arg-strokes (at positions 6 and 8) was to increase the searchability of the compounds (e.g., N-Ac-D-Nal (2) 1, D-pCl-Phe2, D-Trp3, Arg5, D-Tyr6, D-Ala10) LHRH; ED ^ Q = 30 2ug / ml in histamine release; RW Roeske et al., • 5 "Substitution of Arg for Tyr in GNRH antagonists".

Peptides - Structure and Function, ed. C.M. Deber, V. J. Hruby and K. D. Koppie, Pierce Chemical Co.,

Rockford, Illinois, 1985, p. 561). Although this impaired the ability of 35 analogs to degranulate mast cells and release * 4 9/326 histamine, the analog had an increasing number of times the ability to elicit an anaphylactic-type reaction than LHRH with an EDgg of 328 pg / ml in terms of histamine release.

5 Mytts Lys (iPr) is included at various positions in LHRH antagonists. When placed in the 6- and 8-positions with a simultaneous D-pCl-Phe residue in the 2-position, high anti-ovulation potency and decreased histamine release are maintained (e.g., N-Ac-D-Nal (2)). pCl-Phe2, qo in 10 D-Trp, D-Lys (iPr), D-Ala) LHRH; EDj-q = 6.6 pg / ml in histamine release). The ErSåse analog was placed in the 8-position hArg (Et-), whereby the ability of the analog to release his z 1 amine was at the same level (i.e., N-Ac-D-Nal (2), D-oMe-pCl-Phe2, D-Pal). (3) 3, D-Arg6, hArg (Et 2 O) 8, 10 z 15 D-Ala) LHRH (ED 2 q = 4.9 pg / ml in histamine release). However, it can be seen that these analogs are still very effective histamine releasing agents compared to LHRH. (RW Roeske et al., “LHRH Antagonists with Low Histamine Releasing Activity.” LHRH and its Analogs: Contraceptive and Therapeutic Applications, Part 2, edited by BH Vickery and JJ Nestor Jr., MTP Press, Boston, 1987, p. 17). -24).

The 8- and 9-positions have also been identified as the most important substitutable sites to inhibit histamine-25 release, as the Arg-Pro sequence is a frequently observed feature in neuropeptides that cause degranulation of feed cells. Thus, although the 8-position is, on a scientific basis, a crucial position for substitution, a number of analogs currently known have a significant potential for toxicity and other side effects.

This invention relates to novel, highly potent nonapeptide and decapeptide analogs of LHRH that have very low histamine release capacity and that have the sterically hindered guanide-35 nosubstituted arginyl group at the 8-position, with the exception of the Argyl group at the 6-position. The invention relates to myOs compositions containing these compounds.

59 '/ 326

The invention relates to a compound of the formula (I), A-B-C-Ser-D-E-F-G-Pro-J (I) 123 4 5678 9 10 5 or a physiologically acceptable salt thereof.

Replacement of the L-histidyl group at the 2-position of LHRH converts the peptide to an LHRH antagonist. Replacement of the glycyl group at the 6-position in LHRH with one of the groups mentioned in Erna greatly increases the antagonistic effect. The substitutions at the 1-, 2-, 3-, 5-, 7-, and 10-positions shown herein further help to enhance antagonist activity. Sub-substitution at the G 8 position greatly reduces the effect of the analogs te-15 on histamine release when the Arg at the 6-position is replaced by another group, and is crucial for the safety of the analogs.

As indicated above, various abbreviations for common amino acids can be used to facilitate the description of the present invention in a manner generally accepted in the peptide art as recommended by the IUPAC-IUB Commission on Biochemical Nomenclature (Biochemistry 11 (1972) 1726). All peptide sequences mentioned herein are written according to the generally accepted manner in which N-. The 25 terminal amino acid is on the left and the C-terminal amino acid is on the right.

Abbreviations shown refer to L-amino acids, with the exception of asymmetric-centered (achiral) amino acid glycine and, with further exceptions, those naturally occurring or non-naturally occurring amino acids that are achiral or otherwise labeled D or DL.

It should be noted that when J is -NH-C (O) -NH 2, the C-terminal group is an azaglycinamide group.

The abbreviation D-Glu (AA) means an adduct between anisole and a D-Glutenate to form p-meth-6,9326 oxyphenyl ketone (at the C-terminal end of the glutamic acid side chain), i.e. Glu (pMeO-Ph).

Other abbreviations for ErfiSt are also useful in the description of the invention. In this invention, the amino acids of the native LHRH-5 peptide are replaced with amino acids that do not occur in nature. Particularly commonly used are:

Amino acid residue Abbreviation 3- (2-naphthyl) alanyl Nal (2) 10 3- (p-fluorophenyl) alanyl pF-Phe 3- (p-chlorophenyl) alanyl pCl-Phe 3- (3-pyridyl) alanyl Pal (3) N , N'-guanidino (dimethyl) homoarginyl Dmh or hArg (Me) 2 N, Ν'-guanidino (diethyl) homoarginyl Deh or hArgCEt ^ N, N'-guanidino (dipropyl) homoarginyl Dph or hArg (Pr > 2 N, N'-guanidino (diisopropyl) homoarginyl Dih or hArgiiPr ^ N, N'-guanidino (dihexyl) homoarginyl Dhh or hArg (hexyl-> N, N'-guanidino) Homoarginyl Eha or hArgfCl2 ^ N, N'-guanidino (propeno) homoarginyl Pha or hArgiCl · ^) ^ N, N'-guanidinobis (2,2,2-trifluoroethyl) homoarginyl Bth tax hArg (Cl 2 CF 4) ^ 30 NG-ethyl-NG '- (trifluoroethyl) homoarginyl hArgiCH 2 CFg, Et) NG, NG, -2-trifluoromethyl-2,2-difluoroethylhomoarginyl hArg (CH 2 CF 2 CF 3) 35 n- guanidino (ethyl) homoarginyl Meh or hArg (Et) N-guanidino (propyl) homoarginyl Prh or hArg (Pr) 92326 7

Amino AcidsSubject Abbreviation N-guanidino (isopropyl) -homoarginyl Tph or hArg (iPr) N-guanidino (butyl) homo-arginyl Mbh or hArg (Bu) N, N'-guanidino (dicyclohexyl) homoarginyl Dch or hArg (cyclo- hexyl> 2 N-guanidino (heptyl) homo- arginyl Hha or hArg (heptyl) N-guanidino (ethyl) arginyl Mea or Arg (Et) N, N'-guanidino (diisopropyl) arginyl Dia or ArgiiPr N, N'-guanidino (dicyclohexyl) arginyl Dca or Arg (cyclohexyl) -2 3- (3-piperidyl) alanyl 3-Pia 3- (4-piperidyl) alanyl 4-Pia 20 3 - [( NE-methyl) piperid-4-yl] -alanyl Mpa 3 - [(Non-pentyl) piperidin-4-yl] alanyl Ppa 3 - [(N-benzyl) piperid-4-yl] alanyl Bpa

Non-nicotinyl-D-lysyl Lys (Nic) Νε- (3-pyridyl) acetyl-D-lysyl Lys (pyridylacetyl) 30 3- (2,4,6-trimethylphenyl) alanyl Tmp 2,2-diphenylglycyl As used in the Dpg TS, the term "physiologically acceptable salts" means salts which retain the desired biological activity of the parent compound and which do not have any undesirable toxicological effects. Examples of such salts include 9 2326 δ (a) inorganic acids, e.g. acid addition salts formed with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like, as well as organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, fumaric acid, fumaric acid, salts formed with as-corbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids and polygalacturonic acid; base addition salts formed with metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and the like, and an organic cation formed from N, N'-dibenzylethylenediamine or ethylenediamine; and (c) combinations consisting of (a) and (b), for example, zinc tannate salt and the like.

The term "lower alkyl" means a straight or branched chain saturated hydrocarbon group containing from 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl and t-butyl. . "Alkyl having 1 to 6 carbon atoms" includes the same substituents as lower alkyl but additionally contains groups having 5 or 6 carbon atoms, such as, for example, n-pentyl, n-hexyl and branched groups having 5 or 6 carbon atoms. "Alkyl having 1 to 12 carbon atoms" includes hydrocarbon groups having 1 to 12 carbon atoms, e.g. the aforementioned 30 groups Silia with the difference that the groups can contain as many as 12 carbon atoms.

"Fluoroalkyl" means lower alkyl substituted with 1 to 5 fluorine atoms, for example, CF 3 CH 2, CF 3, CF 3 CF 2 CH 2 and the like.

35 "Halogen" means fluorine, chlorine or bromine.

The abbreviation "N-Ac" means an N-acetyl protecting group, i. an acetyl group bound to the amine nitrogen of the terminal amino acid residue, according to a generally accepted designation.

Preferred compounds of the invention are those wherein A is N-Ac-D-Nal (2); B is D-pF-Phe to D-pCl-Phe; C is D-Trp or D-Pal (3); D is Pal (2), Tyr, Arg, Deh, Mbh, Bth or Pha; E is D-Trp, D-Tyr, D-Pal (3), D-Deh, D-Mbh, D-Bth or D-Pha; F is Leu or Phe; G is Deh, Bth, Mbh or Bha; and J is D-AlaNl · or GlyN1 ^.

Even more preferred substitution patterns are those wherein A is N-Ac-D-Nal (2); B is D-pCl-Phe; C is D-Trp or D-Pal (3); D is Tyr, Arg, Deh, Mbh, Bth or Pha; E is D-Trp, D-Pal (3), D-Tyr, D-Deh, D-Mbh, D-Bth or D-Pha; F is Leu; G is Deh, Mbh, Bth or Pha; and J is D-AlaNH2.

A more preferred group of 25 TSh 3 includes three preferred subgroups: 1. When D is Tyr, E may be either (a) one of the hydrophobic groups D-Trp, D-Pal (3), and D-Tyr, but most preferably D-Trp or D-Pal (3); or (b) any of D-Deh, D-Mbh, D-Bth and D-Pha.

2. When D is Arg, E is one of the hydrophobic groups listed in 1 (a), preferably D-Tyr.

3. When D is Deh, Mbh, Bth or Pha, E is most preferably D-Tyr or D-Pal (3), but mytts D-Trp is preferred.

35 In each of the preferred subgroups G is Deh, Mbh,

Bth or Pha. Particularly preferred are Bth and Deh and preferred. lisin Deh.

> · 92326 10

Thus, examples of more preferred substitution patterns are N-Ac-D-Nal (2) -D-pCl-Phe-C-Ser-Tyr-C-Leu-G-Pro-D-AlaNI, where C is D-Trp or D- Pal (3) and G are Deh, Mbh, Bth or Pha; N-Ac-D-Nal (2) -D-pCl-Phe-C-Ser-Tyr-E-Leu-G-5 Pro-D-AlaNH2, where C is D-Trp or D-Pal (3), E is D-Deh, D-Bth, D-Mbh or D-Pha and G is the L-form of E as defined in this paragraph; N-Ac-D-Nal (2) -D-pCl-Phe-C-Ser-Arg-E-Leu-G-Pro-D-AlaNH2, where C is D-Trp or D-Pal (3), E is D-Trp, D-Pal (3) or D-Tyr and G is Deh, 10 Mbh, Bth or Pha; and N-Ac-D-Nal (2) -D-pCl-Phe-C-Ser-D-E-

Leu-G-Pro-D-AlaNH2, where C is D-Trp or D-Pal (3), D and G are each independently Deh, Bth, Mbn or Pha and E is D-Tyr, D-Trp or D- Pal (3).

Other preferred embodiments are N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-E-Leu-G-Pro-D-Ala-NH2, where E is D-Trp or D-Tyr and G is Deh, Bth, Mbh or Pha; N-Ac-D-Nal (2) -D-pCl-Phe-C-Ser-DE-Leu-G-Pro-D-AlaNH2, where D and G are each independently Deh, Bth, Mbh or 20 Pha and C and E is each independently D-Trp or D-Pal (3); and N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu-G-Pro-D-AlaNH2, where G is Deh , Bth, Mbh or Pha.

Examples of preferred embodiments are -25 as follows: • N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu-Deh-Pro -D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu-Bth-Pro-D-AlaNH 2; 30 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -

Leu-Mbh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu-Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-35 Deh-Pro-D-AlaNI ^; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-

Bth-Pro-D-AlaNH0; • z 11 52326 N-Ac-D-Nal (2) -D-pCl-Phs-D-Trp-Ser-Tyr-D-Trp-Leu-Mbh-Pro-D-AlaNH2; U_Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp

Leu-Pha-Pro-D-AlaNH 2; 5 N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Deh-

Leu-Deh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Mbh-Leu-MPH-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Bth-10 Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-PNA-Leu-Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Deh-Leu-Deh-Pro-D-AlaNH 2; ^ N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Mbn-

MDH-Leu-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-3th-Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pha-20 Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Deh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Mbh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -Leu-Mbh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -

Leu-Deh-Pro-D-AlaNH 2; 35 12 92326 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -

Leu-Pha-Pro-D-AlaNH 2; N_Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Leu-Den-Pro-D-AlaNH 2; 5 N-Ac-D-Nal (2) -D-pCl-Phe-D-T rp-Ser-Arg-D-Tyr-

Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Leu-MDH-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-TyΓΙΟ Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Tyr

Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Tyr-Leu-Mbh-Pro-D-AlaNH 2; 15 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Tyr-

Leu-Deh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Tyr-Leu-Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-T rp-Ser-Deh-D-Tyr-Leu-20 Deh-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Mbh-D-Tyr-Leu-Mbh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Bth-D-Tyr-Leu-Bth-Pro-D-AlaNH 2; 25 N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Pha-D-Tyr-Leu-

Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Deh-D-T rp-Leu-Deh-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Mbh-D-Trp-Leu-30 Mbh-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Bth-D-Trp-Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Pha-D-Trp-Leu-Pha-Pro-D-AlaNH 2; 35 13 92326 N-Ac-D-Nal (2) -D-pCl-Phs-D-Pal (3) -Ser-Deh-D-Pal (3) -Le'J-Deh-Pro-D-AlaNh2; N-Ac-D-Nal (2) -D-pCl-pne-D-Pal (3) -Ser-MBN D-Pal (3) -Leu-Pro-MDH-U-AlaNH 2; 5 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-3th-D-Pal (3) -

Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Pha-D-Pal (3) -Leu-Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Deh-D-Nal (2) -10 Leu-Deh-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Mbh-D-Nal (2) -Leu-Mbh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-T rp-Ser-3th-D-Nal (2) -Leu-Bth-Pro-D-AlaNH2; 15 N-Ac-D-Nal (2) -D-pCl-Phe-D-T rp-Ser-Pha-D-Nal (2) -

Leu-Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Pal (3) -D-Pal (3) -Leu-Deh-Pro-D-AlaNH 2; .

N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Pal (3) -20 D-Pal (3) -Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Pal (3) -D-Pal (3) -Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Pal (3) -D-Pal (3) -Leu-Mbh-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Mbh-Leu- 3tn-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Mbh-Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pF-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -30 Leu-Deh-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pF-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -

Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pF-PI ^ E-D-Trp-SeΓ-Tyr-D-TΓp-

Leu-Deh-Pro-D-AlaNH 2; -35 92326 14 N-Ac-D-Nal (2) -D-pF-Phe-D-Trp-Ser-Tyr-D-Trp-

3th-Leu-Pro-D-rtlaNH2; N-Ac-D-Nal (2) -D-pF-Phe-D-Trp-Ser-Tyr-D-Denhardt

Leu-Deh-Pro-D-AlaNH 2; 5 N-Ac-D-Nal (2) -D-pF-Phe-D-T rp-Ser-Tyr-D-Bth-

Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pF-Phe-D-Pal (3) -Ser-Tyr-D-Den-Leu-Deh-Pro-D-AlaNH ^; N-Ac-D-Nal (2) -D-pF-Phe-D-Pal (3) -Ser-Tyr-D-Bth-10 Leu-3th-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pF-Phe-D-Trp-Ser-Arg-D-T rp-Leu-Deh-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pF-Phe-D-Trp-Ser-Arg-D-Trp-Leu-3th-Pro-D-AlaNH 2; 15 N-Ac-D-Nal (2) -D-pF-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -

3th-Leu-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pF-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -Leu-Deh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pF-Phe-D-T rp-Ser-Deh-D-Tyr-Leu-20 Deh-Pro-D-AlaNH2; and N-Ac-D-Nal (2) -D-pF-Phe-D-Trp-Ser-Bth-D-Tyr-Leu-Bth-Pro-D-AlaNH2.

Also included within the scope of this invention are peptides that are not necessarily included in the aforementioned preferred compounds, such as the following: N-Ac-D-Nal (2) -D-pC1-Phe-DT rp-Ser-Tyr-D-Tyr-Leu -Deh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Tyr-Leu-Mbh-Pro-D-AlaNH 2; 30 N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Tyr-

Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Tyr-Leu-Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-35 D-Nal (2) -Leu-Deh-Pro-D-AlaNH2; «·> •» 92326 15 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Nal (2) -Leu-Mbh-Pro-D- Alani ^; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Nal (2) -Leu-Bth-Pro-D-AlaNI ^ · 5 N-Ac -D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Na- (2) -

Leu-Pha-Pro-D-Alani ^; N-Ac-D-Nal (2) -D-AME, pCl-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -Leu-Btn-Pro-D-Alani ^; N-Ac-D-Nal (2) -D-aMe, pCl-Phe-D-Pal (3) -Ser-Tyr-D-10 Pal (3) -Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-AME, pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Glu (AA) -Leu-Bth-Pro-D-AlaNH 2; 15 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-

Glu (AA) -Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Glu (AA) -Leu-Bth-Pro-D-AlaNH2? N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-20 Pal (3) -Leu-HArg (CH2CF2CF3) -Pro-D-AlaNH2; and N-Ac-D-Nal (2) -Dpg-D-Pal (3) -Ser-Tyr-D-Pal (3) -Nal (2) -Bth-Pro-D-AlaNH2.

It is generally preferred that the A, B, C, E and J amino acid residues be in the form of the D-isomer and that the D, F-.25 and G-amino acid residues be in the form of the L-isomer. Stereochemistry should be used unless otherwise specified.

In all of the foregoing embodiments, the compound may be prepared in the form of a corresponding pharmaceutically acceptable salt.

The compounds of the present invention, and in particular the salts thereof, have an excessively potent and long-lasting LHRH antagonist activity.

16

9.1 o L O

The primary measure of the potency of LHRH antagonism is the ability to inhibit ovulation in rats, as determined by A. Corbin and C.W. The methods of the Beatts (Endocrine Res. Commun. 2 (1975) 1).

The ability to induce histamine release from rat peritoneal stem cells in vitro can be mSSrit as described by Sydbom and Terenius (Agents and Action 16 (1985) 269) or Siraganian et al. (Manual of Clinical Immunology, 2nd ed., Ed. N.E.

10 Rose and M. Friedman, Amer. Soc. Microbiol., Washington, D.C., 1980, p. 808).

Other biological assays that can be used to study LHRH antagonists and compounds of this invention include the inhibition of LHRH-induced FSH and LH release in rats in vivo (JA Vilchez-Martinez et al. ., Endocrinology 96 (1975) 1130); (b) inhibition of LH and FSH release by radioimmunological measurement by dispersion culture of anterior pituitary cells (W. Vale et al., Endocrinology 91 (1972) 562); and (c) lowering gonadotropin levels in castrated rats and dogs (Petrie et al., Male Contraception, Harper and Row, Philadelphia, 1985, p. 361).

25 Antagonistic effects and potential uses • é

The following uses result from the antagonistic effect of the compounds described here: - prevention of infertility in females, - prevention or delay of ovulation, 30 - prevention of infertility in males, - diagnostic uses such as the diagnosis of osteoporosis, - other uses described in B Reviews 7 (1986) 115.

II

17 92326

When applying the compounds of this invention, e.g. to inhibit ovulation, the desired subject is administered a compound of the present invention or a composition therefor. These compounds or compositions may be administered in various ways depending on the precise use, e.g. via the oral, parenteral (which includes subcutaneous, intramuscular and its laskimonsisai-administration), vaginally (particularly for hedelmttityksen prevention), rectally, buccally 10 (such as, for example, sublingual), transdermal or intranasal. The most appropriate route in each case depends on the intended use, the active ingredient and the target. The compound or composition may also be administered in a controlled manner using active ingredient depot or injection formulations, which are described in more detail below.

For the foregoing uses, it will generally be expedient to administer the active ingredient in an amount of from about 0.001 to about 5 mg / kg of body weight. The substances are preferably administered to humans at about 0.01-1 mg / kg / day and to non-humans at about 0.1-1 mg / kg / day. This administration may be performed in a single dose, in divided doses, or in a sustained release form for the most effective results. In the case of question 25 for the prevention of heat or pregnancy, the dose is most preferably about 1 to 10 mg / kg and is administered in a single dose.

Parenteral administration generally requires a lower dosage than other methods of administration that are more dependent on absorption.

The invention also relates to compositions comprising as active ingredient a compound of this invention ♦ in admixture with a physiologically acceptable non-toxic carrier. As mentioned above, such compositions may be formulated for parenteral (subcutaneous, intramuscular or intravenous) administration, especially in the form of liquid solutions and suspensions; for use in vaginal or rectal administration particularly in semisolid forms 5, such as creams and suppositories; for oral or buccal administration, especially in the form of tablets or capsules; or for nasal administration, in particular in the form of powders, nasal drops or aerosols.

The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsyl-15, 1970. For parenteral administration. the formulations may contain conventional cytotoxic agents, for example, sterile water or physiological saline, alkylene glycols such as propylene glycol, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalenes and the like. The formulations intended for administration via the vaginal or rectal use, for example, suppositories, may your built-fillers, for example, polyalkylene glycols, petroleum jelly, cocoa butter and the like. Formulations for nasal administration may be solid and may contain as excipients, for example, lactose and dextran, or may be aqueous or non-aqueous solutions intended for administration in the form of nasal drops or metered spray. Typical excipients for buccal administration include sugars, calcium stearate, magnesium stearate, pregelatinized observation, and the like.

Nasal administration of the nona and decapeptides of this invention is particularly preferred. Absorption through the nasal mucosa is enhanced by surfactants

O o 7 O L "s lO LO

pot such as glycocholic acid, cholic acid, tau rocholic acid, cholanoic acid, etocholic acid, deoxycholic acid, chenodeoxycholic acid, dehydrocolic acid and glyco deoxycholic acid.

One or more surfactant acids or salts, but preferably one physiologically acceptable acid salt, may be added to the solution or powder formulation containing the LHRH antagonist. Suitable physiologically acceptable surfactant salts are those which, when used, preserve the improved absorption of the peptide as well as the surfactant properties of the compound and which are not harmful to the subject or otherwise unsuitable. Such salts include, for example, salts obtained from inorganic emulsions, e.g. sodium, ka-15 lithium, lithium, ammonium, calcium, magnesium, aluminum, iron (III) and magnesium (III) salts and the like. Ammonium, potassium, sodium, calcium and magnesium salts are particularly preferred. Salts from physiologically acceptable toxic organic bases include primary, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines), tripropylamine, cyclopamine amines, cyclopamine amines, triethylamines, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, tromethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylamine, ethylenediamine, glycosamine, methylamine, methylamine , salts of piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred toxic organic bases are isopropylamine, diethylamine, ethanolamine, tromethamine, dicyclohexylamine, choline and caffeine.

. · 20 9 '/ ό i 6

More preferably, the surfactant used in the practice of the present invention is an alkali metal salt of glycolic acid, most preferably sodium glycolate.

The surfactant is used in the applications of the present invention to the extent that it enhances the absorption of LHRH peptides more than other surfactants that may enhance the absorption of the peptides in any of the paints. It has been found that often such an amount is 0.2-15% (w / v), more often 0.2-5% (w / v) of solution. The surfactant is preferably present in an amount of about 0.5-4% (w / v), preferably about 1% (w / v) and preferably about 2% (w / v).

Other substances may be added to these formulations, such as preservatives, salts to achieve a tissue-like electrolyte content, and other additives known in the chemistry of nasal formulations. Particularly preferred such other substances are surfactants, preferably nonionic surfactants such as polysorbates, in an amount of 0.1-5% (w / v), more preferably 0.25-2% (w / v) , in concentrations.

It has been found that in order to achieve better solubility and stability, the molar ratio of bile acid to peptide is at least 20: 1, for example at least 25: 1.

It is especially desirable to release the compounds of this invention over a long period of time, for example over a period of weeks to years, from a single administration. Various sustained release depot implants or injectable forms may be utilized. For example, the An-35 dosage form may contain a physiological

II

»· 21 Π r. * 7 ιΛ SL · ^ Ο Ο a highly acceptable toxic salt having a solubility in body fluids of plenl, eslmerklksl (a) a polybasic acid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tartaric acid, tannic acid, pamonic acid, alginic acid, alginic acid, alginic acid - an acid addition salt of polygalacturonic acid or -disulfonic acids, corresponding to polygalacturonic acid; (b) an organic cation of a polyvalent metal cation such as n, N'-dibenzylethylenediamine or ethylenediamine formed by a polyvalent metal cation such as slurry, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium or the like, or for example salt; or (c) a combination of (a) and (b), for example a zinc tannate salt.

In addition, the compounds of this invention, or preferably, their relatively insoluble salts, such as the salts just set forth above, may be formulated into a suitable gel for injection, for example, an aluminum monostearate gel using sesame oil. Particularly preferred salts are zinc salts, zinc tannate salts, pamoate salts and the like.

Another type S sustained release depot formulation suitable for administration as an injection or implant into a tissue could contain the compound or a salt thereof dispersed or encapsulated in a slowly degradable, toxic, non-antigenic polymer such as, for example. polylactic-polyglycolic acid polymer. The compounds or, preferably, their relatively insoluble salts, such as the salts set forth above, may be formulated as cholesterol matrix pellets or silastomer matrix implants or hydrogel or pore capsules, especially when not intended for use. Other sustained release depot and injection formulations, for example liposomes, are well known in the art. Reference is made, for example, to Sustained and Controlled Release Drug Delivery Systems, ed. J.R. Robinson, Marcel Dekker, Inc., New York, 5 1978. In particular, the source of LHRH-type compounds can be found in U.S. Patent 4,010,125. See also C.C. Pitt's article "The controlled delivery of polypeptides including LHRH analogs" in LHRH and its Analogs: Contraceptive and Therapeutic Applications, Part 2, ed. B. H. Vickery and J.J. Nestor, Jr., MTP Press, Boston, 1987, p. 557.

Synthesis of peptides

The polypeptides of the present invention can be synthesized by methods familiar to those skilled in the art of peptide. An excellent summary of many such useful procedures can be found in J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, 1969 and J. Meienhofer, Hormonal Prosteins and Peptides, Volume 2, Aca-20 demic Press, New York, 1973, p. 46, in the case of solid-phase synthesis of peptides, and E. Schroder and K. Lubke, The Peptides, Part 1, Academic Press, New York, 1865, when it comes to classical solution synthesis.

In general, these methods involve the sequential addition of one or more amino acids or an appropriately substituted amino acid to a growing peptide chain. Normally, either the amino group or the carboxyl group of the first amino acid is protected with a suitable protecting group. The protected or derivatized amino acid can then either be attached to an inert solid support or utilize as a solution the next amino acid in the sequence in which the free group (amino or carboxyl group) is suitably protected under conditions suitable for amide bond formation. From this newly linked amino acid residue of its verse 35, the protecting group 92326 23 is removed, and then the next (appropriately protected) amino acid is added, etc. After all amino acids are attached in the correct order, all protecting groups (and any solid moieties) are attached. -5) is removed sequentially or simultaneously to give the final polypeptide. By modifying this general procedure in a simple manner, it is possible to add more than one amino acid at a time to the growing chain, for example a tripept-10 protected peptide and a suitably protected dipeptide (under conditions such that the centers of asymmetry are not racemized) to form protection. peptide.

Preferred Embodiments of the Synthesis A particularly preferred method for preparing the compounds of this invention involves solid phase peptide synthesis.

In this particularly preferred method, the α-amino functional group of the amino acids is protected by acid or 20 female-sensitive groups. Such protecting groups should be stable in properties under peptide bond formation conditions while being easily removed without disrupting the growing peptide chain or racemizing any of the asymmetric centers it contains. Suitable protecting groups include t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, α, α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrile nyl, 2-cyano-t-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl and the like, especially t-butyloxycarbonyl.

Particularly preferred groups for side chain protection in the case of arginine are nitro, p-toluenesulfonyl, 4-methoxybenzenesulfonyl, Cbz, Boc and adaman-35 tyloxycarbonyl; in the case of tyrosine, benzyl, o-bromobenzyloxycarbonyl, 2,6-dichlorobenzyl, isopropyl, cyclohexyl, cyclopentyl and acetyl; in the case of serine, benzyl and tetrahydropyranyl; and in the case of histidine, benzyl, p-toluenesulfonyl and 2,4-dinitrophenyl.

The 5 C-terminal amino acid is attached to a suitable solid support. Suitable solid carriers which can be used in the above synthesis are those which are inert to the reagents and conditions of the stepwise condensation deprotection reactions and which are insoluble in the vehicles used. Suitable solid carriers include chloromethyl-polystyrene-divinylbenzene polymer, hydroxymethylpolystyrene-divinylbenzene polymer and the like, especially a polymer consisting of chloromethylpolystyrene and 1% divinylbenzene. In the special case that the C-terminal residue of the compound is glycinamide, one particularly useful carrier is the benzhydrylaminopolystyrene-divinylbenzene polymer described by P. Rivaille et al., Helv. Chim. Acta 54 20 (1971) 2772. Attachment to a chloromethylpolystyrene-divinylbenzene type resin is accomplished by reacting an N4-protected amino acid, especially a Boc amino acid, with cesium, trimethylammonium, triethylammonium or 1,5-diazabecyclo, As its 5-ene salt or other similar salt in ethanol, acetonitrile, Ν, Ν-dimethylformamide (DMF) or the like, especially as the cesium salt in DMF, at elevated lamp temperature with chloromethyl resin, for example at about 40-60 ° C, preferably about At 50 ° C, about 12-48 hours, preferably about 24 hours. The N, N'-diisopropylcarbodiimide (DIC) / 1-hydroxybenzotriazole (HBT) is coupled to the benzhydrylamine resin by a coupling reaction in a solvent for about 2-24 hours, preferably about 12 hours, such as di-35 in chloromethane or DMF, preferably dichloromethane, at a temperature of about 10-50 ° C, preferably 25 ° C. The incorporation of successive protected amino acids can be accomplished in an automated polypeptide synthesizer known in the art. The removal of the N-protecting groups can be carried out, for example, with a methylene chloride solution of trifluoroacetic acid, a dioxane solution of hydrogen chloride, an acetic acid solution of hydrochloric acid or another strong acid solution, preferably a 50% solution of trifluoroacetic acid in dichloromethane, at approximately ambient temperature. Each protected amino acid is preferably added approximately 2.5 times the excess and the coupling may be carried out in dichloromethane, dichloromethane-DMF or the like, especially dichloromethane, at approximately ambient temperature. The coupling is normally DCC in dichloromethane but may also be N, N * -diisopropylcarbodiimide (DIC) or another carbodiimide, either alone or in combination with HBT, N-hydroxysuccinimide, other N-hydroxyimide or with oxime. Alternatively, protected active esters of amino acids (e.g., nitrophenyl and pentafluorophenyl ester and the like) or symmetrical anhydrides may be used.

At the end of solid phase synthesis, the fully protected polypeptide is cleaved from the resin. When the bond between the polypeptide and the resin support is of the benzyl ester type, cleavage is accomplished by aminolysis in the case of peptides containing proline as the C-terminal amino acid with alkylamine or fluoroalkylamine, or by aminolysis of peptides containing proline. As the C-terminal amino acid in the case of glycine, for example ammonia-methanol or ammonia-ethanol combinations, at a lamp temperature of about 10-50 ° C, preferably about 25 ° C, with a reaction time of about 12-24 hours, preferably about 18 hours 35 hours. Alternatively, the peptide can be cleaved from the resin by transesterification, for example with methanol, followed by aminolysis. At this stage, the protected peptide can be purified by silica gel chromatography. Removal of side chain protecting groups from the poly-5 peptide is accomplished by treating the aminolysis product with, for example, anhydrous liquid hydrogen fluoride in the presence of anisole or another carbonium acceptor, palladium-palladium-hydrogen-trifluoroacetic acid or tris (trifluoroacetate) fluoroacetylacarboxylate, in the presence of a polyvinylpyrrolidone catalyst or by reducing it with sodium in liquid ammonia, preferably liquid hydrogen fluoride and anisole at a temperature of about -10 to + 10 ° C, preferably about 0 ° C, for about 15 minutes at a temperature of about 15 minutes. preferably about 30 minutes. In the case of glycine-terminal amino acids on benzhydrylamine resins, cleavage and deprotection of the resin can be combined into a single step in which the user-20 is favored with liquid hydrogen fluoride and anisole as described above. The deprotected polypeptide is then purified by a series of chromatographic steps using one or all of the following types of steps: with a weakly basic resin in the form of ion exchange chromatography; hydrophobic adsorption chromatography on non-derivatized polystyrene divinylbenzene resin (e.g., with Amberlite XAD); silikageeliadsorptio chromatography; ion exchange chromatography on carboxymethyl-30 cellulose; partition chromatography, for example Sep-hadex G-25 countercurrent distribution; and high performance liquid chromatography (HPLC), especially reverse phase HPLC using column bound phase - packed with octyl or octadecylsilyl silica.

When a racemic amino acid is used at the 1-, 2-, 3- or 6-position, the diastereomeric nonapeptide or decapepto 7 9 cy i. Sj i. O 27 end products are separated and the desired peptide containing the D-amino acid at the appropriate position is isolated. and purified, preferably during the aforementioned chromatographic process.

The preparation of peptides containing the C-terminal azaglycinamide is preferably carried out using classical solution synthesis of peptides and known peptide products. This is described in more detail in Example 3.

Compounds of formula (I) and their physiologically acceptable salts are generally prepared by deprotecting a protected polypeptide and optionally a covalently bonded solid support to provide a compound of formula (I) or a salt thereof; or two fragments of the desired compound of formula (I) are joined to one of the required sequences; or (a) converting a compound of formula (I) into a physiologically acceptable salt; (B) converting a salt of a compound of formula (I) into a physiologically acceptable salt or (c) converting a salt of a compound of formula (I) into a free polypeptide of formula (I).

The following examples are provided to enable those skilled in the art. 25 Ti people are able to get a better understanding of the invention and apply it in practice. They should not be construed as limiting the scope of the invention, but merely as illustrating and illustrating the invention.

Preparation A

3- (2-Naphthyl) -DL-alanine 3- (2-naphthyl) -DL-alanine is prepared by the procedure described in U.S. Patent No. 4,341,767.

The preparation and conversion of N-acetyl-3- (2-naphthyl) -DL-alanine to methyl N-acetyl-3- (2-naphthyl) -DL-alaninate and the separation of the D-isomer are carried out in U.S. Pat. No. 9,232,268. 4,341,767.

Preparation B

Benzyl N, N-benzyloxycarbonyl N, N'-guanidinodi-5-isopropyl-D-homoargininate toluenesulfonate

To a mixture of 5.24 g of benzyl-N-benzo-carbonyl-D-lysinate toluenesulfonate (B. Bezus and L. Zervas, J. Am. Soc. 83 (1961) 719) and 1.72 ml of di-iso -propylethylamine in 60 ml of dioxane, 1.89 g of N, N'-diisopropylcarbodiimide were added. The reaction mixture was stirred at 100 ° C for 6 hours and cooled to room temperature and evaporated to give a solid. The solid was suspended in 20 ml of warm DMF, the suspension was filtered to remove N, N'-diisopropylurea and the filtrate was evaporated to give a solid. Benzyl N, N'-benzyloxycarbonyl-N, N'-guanidinodiisopropyl-D-homoargininate toluenesulfonate was obtained as a white solid by crystallization from a 1M mixture of methanol and ethyl acetate; 20 DEG C. = 7.26 ° (c = 0.3, MeOH).

Accordingly, using the procedure described above but replacing N, N'-diisopropylcarbodiimide with N, N1-dicyclohexylcarbodiimide; N, N'-di (n-hexyl) carbodiimide; * '25 N, N'-diethylcarbodiimide; N, N'-di (n-propyl) carbodiimide; N-diisopropylcarbodiimide; N-propyl carbodiimide; N- (n-butyl) carbodiimide? . N, N'-di- (n-butyl) carbodiimide; : Ν, Ν'-diisobutylcarbodiimide? Ν, Ν'-di (n-pentyl) carbodiimide; N, N1-di-isopentyylikarbodi-imide; N, N1-diphenylcarbodiimide-imide; 35 N, N'-ditolylcarbodiimide or 92326 29

1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride or the like gives benzyl N, N'-benzyloxycarbonyl-Ν, Ν'-guanidinodicyclohexyl-D-homoarginate, = 8.07 ° (c = 0 , 9, MeOH), benzyl-N

5 benzyloxycarbonyl-N, N'-guanidinodietyl-D-homoargininate, benzyl-N-benzyloxycarbonyl-N-N'-guanidinodi- (n-propyl) -D-homoargininate, 8,08.07 ° (c = 0 , 9, MeOH), benzyl N * · -benzyloxycarbonyl-N-guanidino- (n-propyl) -D-homoargininate, benzyl-N * -benzyloxycarbonyl-N-guanidino- (n-butyl) -D- homoargininate, benzyl N * -benzyloxycarbonyl-N, N'-guanidinodi- (n-butyl) -D-homoargininate, benzyl-N * -benzyloxycarbonyl-N, N-guanidinodiiso-15-butyl-D-homoargininate, benzyl- N * -benzyloxycarbonyl-N, N'-guanidinodi- (n-pentyl) -D-homoargininate, benzyl-N * -benzyloxycarbonyl-N, N'-guanidinodiphenyl-D-homoargininate, benzyl-N * -benzyloxycarbonyl- N, N'-guanidinodimethyl-D-homoargininate, benzyl-N * -benzyloxycarbonyl-N, N'-guanidinodi- (n-hexyl) -D-homoargininate and benzyl-N * -benzyloxycarbonyl-N, N1-guanidinodi- iso-: 25 propyl D-argininate, [α] 20 D = -10.5 ° (c = 0.5, MeOH), benzene sulfonate. Similarly, by substituting benzyl N * benzyloxycarbonyl D-orninate for D-lysinate, the corresponding arginine analogs can be obtained as their toluenesulfonate salts.

30 Preparation C

^^ Q |

Benzyl N-benzyloxycarbonyl N, N-etheno-D-homoargininate

To a mixture of 15 mL of toluene and 15 mL of t-BuOH were added 2.71 g of benzyl N * -benzyloxycarbon-35 carbonyl lysinate and 1.46 g of 2-methylthioimidazoline-92,9326 hydroiodide (available from Aldrich). The pH of the mixture was adjusted to approximately 8 by the addition of diisopropylethylamine, and the solution was refluxed for 24 hours.

The solution was stabilized under reduced pressure, and the portion was packed into a silica gel column (250 mg). The column was eluted using a C 1 -C 4 -MeOH mixture gradient from 19: 1 to 7: 3. The product-containing fractions were identified by TLC, combined and evaporated to dryness to give 2.9 g of a white foam.

A 2 g portion of the above product was dissolved in 50 mL of EtOH containing 0.8 g of a catalyst containing 10% Pd on carbon. The solution was stirred at Η2 »· η for less than 8 hours. The mixture was filtered through celite (diatomaceous earth) and the filtrate evaporated to dryness to give 1.2 g of N, N-ethanohomoarginine as a white foam.

In a similar manner but using S-methyl-3,4,5,6-tetrahydro-2-pyrimidinethiol (Aldrich) G G1 as the guanylating agent, N, N-propanohomoarginine was obtained as a white foam.

Similarly, but using S-methylbis- (2,2,2-trifluoroethyl) -thouronium iodide G G1 as the guanylating agent, N, N-bis- (trifluoroethyl) -homoarginine (Bth) was obtained as a white foam.

Preparation D

Ν '** t-Butyloxycarbonyl-N, N'-guanidinodiisopropyl D-homoarginine toluenesulfonate This preparation example illustrates the preparation of N, N'-guanidinodi-substituted D-homoarginines from their toluene sulfonate precursors.

A mixture of benzyl N, N'-benzyloxycarbonyl-carbonyl N, N'-guanidinodiisopropyl-D-homoargininate toluenesulfonate (3.25 g) and 100 mg of a catalyst containing 10% Pd on carbon was added. per ml of glacial acetic acid, was treated with hydrogen gas at atmospheric pressure for 35 4 hours. The catalyst was filtered off

II

31

o t ', "7 S

The celite was evaporated to give a solid N, N'-guanidinodiisopropyl-D-homoarginine toluenesulfonate. To a solution of the compound (2.13 g) in 60 ml of a dioxane-water mixture (1: 1) was added 10 ml of 1N sodium hydroxide and 0.4 g of magnesium oxide. Then, 1.1 g of di- (t-butyl) dicarbonate was added to the mixture, and it was stirred at room temperature for 2 hours. The magnesium salt was filtered off and the filtrate was concentrated under reduced pressure. The basic solution was washed with ethanol and then adjusted to pH 2.5 with sodium sulfate. The acidic aqueous solution was extracted with ethyl acetate, and the ethyl acetate solution was dried over magnesium sulfate. The desiccant was removed by filtration and the filtrate was evaporated. Crystallization from ethyl acetate-hexane-15 gave t-butyloxycarbonyl-N, N1-guanidino diisopropyl-D-homoarginine toluenesulfonate.

By proceeding in the same manner but using appropriate toluenesulfonate precursors, other Ν ° ί-t-butyloxycarbonyl-N, N'-guanidino di-substituted D-homoarginine or D-arginine compounds can be prepared.

Preparation E

t-butyloxycarbonyl-3- [4 '- (1'-propylpiperidyl) -γ-D-alanine To 400 ml of absolute ethanol was added 4.6 g of sodium metal and the mixture was heated. To the resulting sodium ethoxide solution were added 21.7 g of diethyl (acetamidomalonate) and 16.4 g of 4-picolyl chloride hydrochloride. 30 chloride (Aldrich Chemical Co). The reaction mixture was heated at 100 ° C for 4 hours and cooled, filtered and stabilized under reduced pressure. The mixture was applied to a silica gel column in methylene chloride-methanol (19: 1) and eluted with the same mixture. The product was localized to a fast moving UV-positive spot on a silica gel thin layer chromatogram (methylene chloride-methanol 19: 1 as eluent). The combined fractions were concentrated to give the product.

The product of the previous paragraph was dissolved in 200 5 ml of ethanol, and this solution was treated with a solution of 2.72 g of sodium hydroxide in 40 ml of water at 50 ° C for 6 hours. The solution was acidified with 12 mL of 6 N HCl, evaporated to dryness, and the residue was stirred in 200 mL of dioxane. The suspension was filtered and 10 filtrates were refluxed for 2 hours. The solution was cooled and evaporated to dryness to give ethyl N, N-Cetyl-3- (4-pyridyl) -DL-alanine as a white solid.

A portion of this N-acetyl ester was redissolved by treating it with 200 ml of subtilisin Carls-15 berg (Sigma Chem. Co., Protease VIII) in a mixture containing 300 ml of dimethyl sulfoxide and 400 ml of 0.01 M KCl, (pH 7.2 ). After 6 hours, redissolution was complete. The solution was diluted with 400 ml of water and extracted with four 750 ml portions of ethyl acetate. The organic layers were combined and dried over magnesium sulfate and concentrated to give ethyl N, N-acetyl-S- (4-pyridyl) -3-alaninate as an oil.

The oil was treated with 1.22 g of n-propyl bromide in 50 ml of ethanol, after which the solution was evaporated to dryness to give ethyl-N, N-acetyl-3- (1-propylpyridinium-4-yl) - D-alaninate bromide as a white hygroscopic solid.

This white solid was dissolved in 200 ml of ethanol and reduced under a hydrogen atmosphere using 100 mg of a combination of 10% Pd on carbon as a catalyst. After 18 hours of reduction, the catalyst was filtered off and the solution was evaporated to give ethyl -acetyl 3- [4 '- (1'-propyl-piperidyl) -β-D-alaninate as a tan solid 35. The free acid was prepared by refluxing ethyl acetate.

II

92326 ester in 100 mL of 6 H HCl for 4 hours to give 3- [4 '- (1'-piperidyl) -7-D-alanine as a white solid.

The free acid was dissolved in 100 ml of a dioxane-water-5 mixture (1: 1), and 2 g of di- (t-butyl) dicarbonate was added to the solution. The pH was maintained at 9 with a pH statate by the addition of 1 N NaOH. After 2 hours, the reaction mixture was concentrated under reduced pressure and washed with 100 mL of ethyl ether, and the aqueous layer was applied to an Amberlite XAD-2 resin-10 column (hydrophobic resin). The column was eluted with 250 ml of water and then with 250 ml of ethanol-water (1: 1). The ethanol eluates were combined and evaporated to dryness to give N-t-butyloxycarbonyl-3- [41- (1'-propylpiperidyl) 7-D-alanine as a white solid.

By proceeding in the same manner but substituting 3-picolyl chloride hydrochloride for 4-picolyl chloride hydrochloride, t-butyloxycarbonyl-3- [3 '- (1'-propylpiperididyl) -7-D-alanine is prepared.

20 Preparation F

Boc-Gly-O resin 4.9 g of Boc-glycine was dissolved in a mixture of 50 ml of ethanol and 50 ml of distilled water. The pH of the solution was adjusted to 7 with aqueous cesium bicarbonate. 25 solution. The solvent was then evaporated under reduced pressure.

After drying under reduced pressure for 18 hours, the residue was dissolved in 150 ml of dry DMF. To the solution was added 25 g of chloromethylated poly-styrene resin containing 1% divinylbenzene (equivalent to 25 mmol of Merrifield * chloride). The mixture was shaken at 50 ° C for 24 hours, filtered, and the resin was then washed successively with DMF, water and ethanol. The resin was dried under reduced pressure for 3 days to give 28.34 g of Boc-Gly-O resin.

34 9 2 3 2 6

Preparation G

A. S-Methyl-3,4,5,6-tetrahydro-2-pyrimidine thiol hydroiodide

To a solution of 23.24 g of 3,4,5,6-tetrahydro-5 2-pyriraidinthiol in 175 mL of MeOH was added 15.57 mL of Mel, and the mixture was refluxed for 1.5 hours. The solvent was evaporated under reduced pressure and the portion was suspended in diethyl ether. The precipitate was filtered off and dried under reduced pressure to give 51.4 g of S-methyl-3,4,4,6-tetrahydro-2-pyrimidinethiol as white crystals.

B. N, N-t-Butyloxycarbonyl-N, N-propeno-L-homoarginine S-methyl-3,4,5,6-tetrahydro-2-pyrimidinethiol was obtained from its 51.4 g of its HI salt by partitioning. Between CH 2 Cl 2 (300 mL) and 4 N NaOH (50 mL). The resulting CH 2 Cl 2 solution was stabilized to a volume of about 100 mL, and about 100 mL of EtOH was added. A solution of S-methyl-3,4,5,6-tetrahydro-2-pyrimidinothiol was added dropwise at 60 ° C to a solution of 24 g of lysine hydrochloride in 66 ml of 2 N NaOH. Stirring was continued overnight at 60 ° C under nitrogen. 10.78 g of additional HI salt was decomposed with 15 mL of 4 N NaOH, extracted with 90% CH 2 Cl 2, and added dropwise with stirring for another 24 h at 60 ° C, during which time the reaction: 25 went to maximum roughly complete.

The reaction mixture was washed with two portions of EtOAc, and the aqueous layer containing Pha was diluted with 200 mL of dioxane and 200 mL of water. The solution was cooled to 0 ° C before 33 mg of di (t-butyl) -30 dicarbonate and 6 g of MgO were added. An additional 4 g of MgO and an additional 22 g of di- (t-butyl) dicarbonate made the reaction complete.

The MgO was separated by filtration through celite, and the filtrate was stabilized under reduced pressure to half its volume.

35 JaannSs was washed twice with diethyl ether before

II

35 9 2 3 2 6 onto a silica gel column (750 g of silica gel packed in a column in CH 2 Cl 2). The column was washed with 5 liters of CH 2 Cl 2. and eluted with CH 2 Cl 2 containing 10% water (total 2 liters), and further with CH 2 CN containing 20% water (total 5 liters). The product fractions were localized by thin layer chromatography on silica gel plates (CH 3 CN-HOAc-H 2 O 4: 1: 1). The product fractions were combined and evaporated to give 5.0 g of product as a white foam, m.p. 96 ° C and 10 = 16.1 ° (c = 0.54, MeOH), and an additional 15 g of a slightly crude oil.

In a similar manner but using the appropriate S-methyl-N, N1-dialkylthiouronium hydroiodides, the corresponding t-butyloxycarbonyl-N, N-dialkylhomo-15 arginines are obtained.

Example 1 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-hArg (CH2CF3) 2 ~ D-Tyr-Leu-hArg (CH2CF3) 2 ~ Pro-D- Ala-NH2 The title compound is shown in this specification also as N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Bth-D-Tyr-Leu-Bth- Pro-D-Ala-NH 2, respectively.

0.758 g (0.5 mmol) of benzhydrylamino-polystyrene resin (Peninsula Labs., 0.66 mmol / g) was charged to the reaction vessel of the Beckman 990 Peptide Synthesizer.

The first amino acid was added by adding 0.284 g

Boc-D-Ala-OH, 0.202 g Hbt. and 3 ml of 0.5 M diisopropylcarbodiimide. After a 3 hour addition period and washing of the resin, other amino acids were added sequentially to the tShS resin according to the synthesis program as follows: Step 1 CH 2 Cl 2 ~ wash once 1.5 min 2 deprotection with CF 3 CO 2 H-CH 2 Cl 2 (1: 1) once 1.5 min 3 protection removal with cf3co2h-ch2ci2-35 (1.1) once 30 min 36 92326 4 Cl2Cl2 wash three times 1.5 min 5 triethylamine-Cl2C2 mixture (1: 1) twice 1.5 min 6 CH2Cl2 washing three times for 1.5 min 7 N -Roc-amino acid dissolved in 5 ° C-CH 2 -DFF mixture (1: 1) once with 8 N, N'-diisopropylcarbodiimide solution (0.5 M) once with 9 Cl 2 Cl 2 rinse and storage, addition once addition reaction 2 hours 10 CH 2 Cl 2 rinse once 1.5 min 11 CH 2 Cl 2 wash three times 1.5 min 12 ethanol wash three times 1.5 min 15 13 CH 2 Cl 2 wash three times 1.5 min

Steps 1-13 terminate the single amino acid addition cycle, and the completion of the reaction can be checked by the nin-hydrine method / E. Kaiser et al., Anal. Biochem.

34 (1970) 595J.

Amino acids were sequentially coupled to the resin using a 2.0-3.0-fold molar excess of each protected amino acid as well as DIC. The resin was treated during subsequent coupling cycles with 0.269 g of Boc-Pro-OH, 0.610 g of Boc-Bth-OH, 0.311 g of Boc-Leu-OH-Cl 2, 0.375 g : 11a Boc-D-Tyr-OH, 0.610 g: 11a Boc-Bth-OH, 0.380 g: 11a Boc-Ser (benzyl) -OH,; 0.320 g of Boc-D-Pal (3) -OH, 0.390 g of Boc-D-pCl-Phe-OH, 0.400 g of Boc-D-Nal (2) -OH and 2.5 ml of acetic anhydride.

The resin was removed from the reaction vessel, washed with CH 2 Cl 2 and dried under reduced pressure to give 1.43 g.

II

92326 37 protected polypeptide-resin combination. The peptide was deprotected and removed from the resin by treatment with a combination of 25 mL of aqueous liquid HF. With 2.5 ml of anisole (acceptor) in a Kel-5 F reaction vessel at 0 ° C for 1 hour, the HF was evaporated under reduced pressure to leave N-Ac-D-Nal (2) -D-pCl-Phe -D-Pal (3) -Ser-hArg (CG2CF3) 2-D-Tyr-Leu-hArg (CH2CF3) 2-Pro-D-Ala-NH2 (as its HF salt) was washed with ether. The portion was then extracted with glacial acetic acid (3 x 30 mL). The acetic acid extraction solution 10 was evaporated to dryness. The crude material was converted to the acetate salt by applying it in water to an acetate-converted AG3 column (weakly basic tertiary amine resin 1 Hpi. Freeze-drying of the eluate gave 0.5 g of crude peptide acetate salt as a white solid.

The crude peptide was purified by HPLC on a 2.5 x 100 cm Licroprep RP-18 column (25.40 μm) equilibrated with 0.1% CF 3 CO 2 in CH 2 CN-H 2 O (1: 1) with a buffer of 20 (pH 2.5). The UV-absorbing (280 nm) overhead peak, which eluted at about 2 times the column volume of eluate, was collected and evaporated to dryness and freeze-dried three times from distilled water to give 64 mg of pure N-Ac. -D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-hArg (CG2CF3) 2- D-Tyr-Leu-hArg (CH2CF3) 2-Pro-D-Ala-NH2 δ FCl 3 = -17.96 ° (c = 0.4, HOAc).

B. By proceeding in the same manner but using the appropriate amino acids in place of the 30 amino acids A, B, C, D, E, F, G and J, the corresponding decapeptides were prepared as exemplified. keja alia: 38 9/326 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu-Den-Pro-D- AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Mbh-D-Pal (3) -Leu-Mbh-Pro-D-AlaNH 2; 5 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Pal (3) -D-Pal (3) -Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -Leu-Deb-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -10 L9u-3tn-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Pne-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu-Mbh-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Pbe-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu-Pha-Pro-D-AlaNH2; 15 N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-

3th-Leu-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Ls'J-Deh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-20 Leu-Bth-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-T rp-Ser-Arg-D-Trp-Leu-Pna-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -Leu-PNA-Pro-D-AlaNH 2; * 25 N-Ac-D-Nal (2) -D-pCl-Phe-D-T rp-Ser-Tyr-D-Btn-

Leu-Bth-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-PNA-Leu-Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Deh-; 30 Leu-Den-Prb-D-AlaNH2; and: N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-3th-

Leu-Bth-Pro-D-AlaNH ^.

C. By proceeding in the same manner but replacing the 35 said amino acids with the appropriate A-, B-, C-, D-, E-,. The corresponding decapeptides are prepared with amino acids F, G and J, examples of which are given below: li 92326 39 N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr- D-Trp-Leu-Deh-Pro-D-AlaNH ^; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-MPH-Pro-D-AlaNH ^; 5 N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-

Leu-Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Tyr-D-Den-Leu-Deh-Pro-D-AlaNh 2; N-Ac-D-Nal (2) -D-pCl-Pne-D-Trp-Ser-Tyr-D-Mbn-Leu-Moh-Prc-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Mbh-Leu-Mbh-Prc-D-Ala NH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pha- ^ Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Pbe-D-Trp-Ser-Arg-D-Trp-Leu-Nbh-Prc-D-Ala NH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -Leu-Bth-Prc-D-AlaNH2; 2o N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -Leu-Mbn-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr

Den-Leu-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-25 Leu-Bth-Pro-D-AlaNH2; : N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-

Leu-Mbh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Le u-Ph a -Pr o-D-Ala NH2; 30 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Tyr-

Leu-Bth-Prc-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Tyr-Leu-Mbh-Prc-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Tyr-35 Leu-Deh-Pro-D-AlaNH2; q o 3 9 6

40 hours

N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Tyr-Leu-Pha-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Deh-D-Tyr-Leu-Deh-Pro-D-AlaNH 2; 5 N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Mbh-D-Tyr-Leu-

Mbh-Pro-D-AlaNH9; N_Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-3tn-D-Tyr-Lej-3tn-Pro-D-AlaNH2; N -Ac-D-Na 1 (2) -D-pCl-Pne-D-Trp-Ser-Pna-D-Ty r-Leu- 10

Pha-Pro-D-AlaNH ^; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Deh-D-T rp-Leu-Deh-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Mbh-D-Trp-Leu-1 Mbn-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-3th-D-Trp-Leu-

Btn-Pro-D-AlaNH ^; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Pha-D-T rp-Leu-Pha-Pro-D-AlaNH 2; 2Q N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Deh-D-Pal (3) -

Leu-Deh-Pro-D-AlaNH ^; N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Mbh-D-Pal (3) -

Leu-Mbh-Pro-D-AlaNH 2;

N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-3tn-D-Pal (3) - «25 Leu-Bth-Pro-D-AlaNh ^ J

N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Pha-D-Pal (3) -Leu-Pha-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Deh-D-Nal (2) -Leu-Deh-Pro-D-AlaNH2 * j 2q N-Ac-D- Nal (2) -D-pCl-Phe-D-Trp-Ser-Moh-D-Nal (2) - *: Leu-Mbh-Pro-D-AlaNH2; N-Ac-D-Nal (2) -D-pCl-PBE-D-Trp-Ser-3th-D-Nal (2) -Leu-3th-Pro-D-AlaNH 2; N-Ac-D-Nal (2) -D-pCl-Phe-D-Trp-Ser-Pha-D-Nal (2) -35 Leu-Pha-Pro-D-AlaNH2; li 41 52326 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Pal (3) -D-Pal (3) -Leu-Deh-Pro-D-AlaNH2 ; N_Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Pal (3) -D-Pal (3) -Leu-Bth-Pro-D-AlaNH 2; 5 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Pal (3) -D-Pal (3) -Leu-Pha-Pro-D-AlaNH2; N_Ac-D-Nal (2) -O-pCl-Phe-D-Pal (3) -Ser-Pal (3) -0-3al (3) -Leu-Mpn-Pro-D-AlaNH2.

Example 2 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-hArg (CH2CF3) 2 ~ D-Tyr-Leu-hArg (CH2CF2) 2 ~ Pro-NHet In the synthesis of analogs containing C-terminal Pro-NHCH2CH2, a program identical to the synthesis program described in Example 1 was used. 0.71 g was placed in the reaction vessel of a Beckman 990-15 synthesizer

Boc-Pro-O resin prepared by reacting a 1.3-fold moderate excess of the dry cesium salt of Boc-Pro-OH with a chloromethylpolystyrene resin containing 1% divinylbenzene (Lab Systems, Inc.).

The amount of Boc-Pro-O resin used contained 0.5 mmol of proline.

Amino acids were sequentially coupled to the resin using a 2.0-3.0-fold molar excess of each protected amino acid and DIC. The resin was thus allowed to react during successive addition cycles: '25 with 0.610 g of Boc-Bth-OH, with 0.311 g of Boc-Leu-OH * H 2 O, with 0.375 g of Boc-D-Tyr-OH with 0.610 g of Boc-Bth-OH, with 0.380 g of Boc-Ser (benzyl) -OH, with 0.320 g of Boc-D-Pal (3) -OH with 0.390 g of Boc-D-pCl-Phe-OH, with 0.400 ml of Boc-D-Nal (2) -OH and with 2.5 ml of acetic anhydride.

The resin was removed from the reaction vessel, washed with CH 2 Cl 2 and dried under reduced pressure to give 1.5 g of the protected polypeptide-resin combination.

92326 by lysis in 25 ml of ethylamine at 2 ° C for a reaction time of 18 hours. The ethylamine was allowed to evaporate and the resin was extracted with methanol. The methanol was evaporated, with 2.5 ml of anisole and 25 ml of redistilled (from CoF) anhydrous liquid HF in a Kel-F reaction vessel at 0 ° C for 1 hour. The HF was evaporated under reduced pressure and the residue was washed with ether. The residue was dissolved in 2 M acetic acid and converted to the acetate salt by passing it through a pad of 10 AC3 columns converted to the acetate form in water. Freeze-drying of the eluate gave 0.5 g of the crude peptide acetate salt as a white solid. Final purification was performed on an HPLC column 2.5 x 100 mm in size containing 40-50 octadecylsilylated silica (Merck, Lichro-15 prep C-g) using 50% CH 2 CN as eluent. a (0.1% CF 2 CO 2 H). Freeze-drying of the product from water gave 70 mg of N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-hArg- (CH2CF2-D-Tyr-Leu-hArg (CH2CF3) 2- ProNHEt as a white powder.

By proceeding in the same manner but using the required protected amino acids, the following compounds are prepared: N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu -Bth-Pro-NHEt, *; 25 N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu-

Mbh-Pro-NHEt, N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu-Pha-Pro-NHEt, N -Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) -Leu-30 Deh-Pro-NHEt, · 'N-Ac-D -Nal (2) -D-pCl-Phe-D-Trp-Ser-Arg-D-Nal (2) -Leu- 3th-Pro-NHEt.

By repeating the above cleavage and replacing the ethylamine with a stoichiometric amount of methyl or propylamine, the corresponding methyl or propyl amides of the above-mentioned nonapeptides are obtained.

Example 3

Compounds of formula (I) may be prepared by myds classical solution synthesis.

For example, the following procedure can be used to join two pentapeptide fragments: N-Ac-D-Nal (2) -Dp-Cl-Phe-Boc-Den-Leu-Den-Pro-D-Ala-NH2 10 Q-? Al (3) -Ser-Tyr-OMs (1) (2) -. v - H-D-Deh-Leu-Deh-Pro-D-Ala-Ny 'f N-Ac-D-Nal (2) -D-p-Cl-Phe-15 D-Pal (3) -Ser-Tyr-N3

V

H-D-deH

Leu-Deh-Pro-AzaGlyNH2 (3) -r N-Ac-D-Nal (2) -Dp-Cl-Phe-D-Pal (3) -Ser-Tyr-D-Deh-Leu-Oeh-Pro- D-Ala-NH? 20 ^

Joining of individual fragments can be accomplished by the acylazide method (J. Honzel et al.,

Coll. Czech. Chem. Commun. 26 (1971) 2333), a DCC / HBT ligand, a diphenylphosphoryl azide coupling, or some other non-racemic fragment coupling technique. Compound (2) can be prepared as described above, and compound (1) can be prepared by methods similar to those described in Example 1. Compound (3) is prepared from compound (2) by removing Cbz by hydrogenation, followed by the addition of N-Boc-N, N * -guanidinodiethyl-D-homoarginine to a DCC-HBT combination or other excipient known in the art. using.

The fragments to be joined in this way are often peptides or amino acids. Alternatively, the N-tert-92326 44 terminal nonapeptide may be prepared by a solid phase or solution method and then coupled to an alkylamine or semicarbazide hydrochloride by dicyclohexylcarbodiimide and hydroxybenzotriazole or by any other coupling method.

Example 4

Preparation of salts A. Solution containing 0.1 g (N-Ac-D-Nal (2) x, 10 D-pCl-Phe2, D-Pal (3) 3.8, Btn8, D-Ala ") The hydrogen fluoride disalt salt of LHRH (i.e. Example 1) dissolved in 50 ml of water was counted on a column containing 50 g of Dowex 3-anion exchange resin previously equilibrated with acetic acid and washed with deionized water.

The column was eluted with deionized water and the eluate was freeze-dried to give the corresponding N-Ac-D-Nal (2) ^, 9 OCOT Λ D-pCl-Phe “, D-Pal (3) ', Bth, D-Ala ) Acetic acid salt of LHRH.

By repeating the above procedure but replacing acetic acid with other acids in equilibrating the resin, for example, corresponding salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, benzoic acid and the like can be prepared.

· * 25 Similarly, other acid addition salts of the peptides analogous to LHRH disclosed in this specification can be prepared.

B. Salts with low water solubility can be prepared from water by precipitation using the desired acid. For example: »« ·· Zinc tannate salt - Solution containing 10 mg (N-Ac-D-

Nal (2) 1, D-pCl-Phe2, D-Pal (3) 3,6, Bth8, D-Ala10) acetic acid salt of LHRH in 0.1 ml of water was treated with a solution containing 8 mg of tannic acid in 0.08 ml in 0.25 M 35 NaOH. A solution of 5 mg of ZnSO 4 -7H 2 O in 0.1 mL of water was immediately added to the solution of the LHRH analog.

The resulting suspension was diluted with 1 ml of water, and the precipitate was separated by centrifugation. The supernatant was removed by decantation, and the residue was washed twice with 5 ml of separate water by centrifugation of the precipitate and decantation of the supernatant. The precipitate was dried under reduced pressure to give 15 mg of the zinc tannate mixed salt of the above-mentioned LHRH analog.

Pamoate salt - To a solution containing 50 mg of the acetic acid salt of 10 (N-Ac-D-Nal (2) 1, D-pCl-Phe2, D-Pal (3) 3'6, Bth8, D-Ala10) -LHRH in a mixture , containing 1.6 ml of ethanol and 0.1 ml of 0.25 M NaOH, a solution of 11 mg of pamonic acid in 0.3 ml of 0.25 M NaOH was added. The solvents were removed under reduced pressure, the residue was suspended in 2 ml of water, the suspension was centrifuged, and the supernatant was decanted. The precipitate was washed with 1.5 ml of water and centrifuged, and the supernatant was decanted. The precipitate was dried under reduced pressure to give 54 mg of the pamoate salt of the above-mentioned LHRH analog.

20 Similarly, other salts with poor water solubility can be prepared.

C. Preparation of acid addition salt from free peptide

To a solution of 50 mg (N-Ac-D-Nal (2) 2, D-pCl-Phe 2, D-Pal (3) 3,6, Bth 8, D-Ala 10) LHRH as a free base was added 30 ml of 1 N acetic acid. The resulting solution was lyophilized to give 50 mg of the acetic acid salt of the above-mentioned LHRH analog.

Similarly, substitution of acetic acid with other acids (in stoichiometrically equivalent amounts relative to the peptide) provides other acid addition salts of the peptides disclosed herein, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and nitric acid.

35 D. Preparation of a salt formed by a metal cation, for example a zinc salt: To a solution containing 50 mg of N-Ac-D-Nal (2) 46 92326 D-pCl-Phe2, D-Pal (3) 3'6, Bth8, D-Ala10) Acetic acid salt of LHRH in a mixture of 0.4 ml of 0.25 M NaOH, 0.3 ml of water and 1 ml of ethanol was added to a solution of 15 mg of ZnSO 4. ^ 0 in 0.2 ml of water. The precipitate was centrifuged and the supernatant was separated by decantation. The precipitate was washed with 1 ml of water by centrifugation and decantation of the supernatant. The precipitate was dried under reduced pressure to give the above zinc salt of said LHRH analog.

Salts can be prepared in a similar manner with other polyvalent cations, for example, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and the like.

Example 5 Conversion of salts to free emM

Solution containing 50 mg of acetic acid salt of LHRH (N-Ac-D-Nal (2) *, D-pCl-Phe2, D-Pal (3) 3'6, Bth8, D-Ala10) in 25 ml: water was lowered onto a column containing 50 g of Dowex 1 (a strongly basic ammonium anion exchange resin) equilibrated with NaOH solution to give a hydroxide counterion. The column was eluted with 150 ml of water, and the eluate was lyophilized to give 45 mg of the corresponding polypeptide as a free mother.

Other acid addition salts of the peptide compounds disclosed herein may be converted to the free sow in a similar manner.

Characterizing

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Table 2 Amino acid analyzes 15 (RS-26334): Ser 0.96 (1); Pro 0.96 (1); Ala 0.98 (1); Leu 0.95 (1); Tyr 1.07 (1); Lys 5 0.09 (0); NH3 1.82 (1); Trp 2.12 (2); p-Cl-Phe 1.00 (1); hrtrg (CH 2 CF 3) 2 0.89 (1); Nal (2) 1.09 (1).

21 (RS-26126): Ser 1.01 (1); Pro 0.99 (1); Ala 0.98 and n - (1); Leu 0.95 (1); Lys 0.20 (1); NH3 1.27 (1); Arg 1.08 (1); Pal (3) 2.10 (2); p-Cl-Phe 1.11 (1); hArg (CH 2 CF 3) 0.79 (1); Nal (2) 1.02 (1).

15 2_2 (RS-26906): Ser 1.03 (1); Pro 0.97 (1); Ala 0.98 (1); Leu 0.90 (1); NH3 2.25 (1); Trp 2.30 (2); Arg 1.12 (1); p-Cl-Phe 1.03 (1); hArg (CH 2 CF 3) 2 0.99 (1);

Nal (2) 1.12 (1).

20 r 31 (RS-64736): Ser 0.84 (1); Pro 0.89 (1); Ala 0.99 (1); Leu 1.01 (1); Lys 1.05 (1); ΝΗ? 0.91 (1); hArg (Et2) 1.04 (1); Pal (3); 25 2.42 (2); p-F-Phe 1.21 (1); Nal (2) 1.13 (1).

32 (RS-64802): Ser 0.92 (1); Pro 0.95 (1); Area 1.01 (1); Leu 0.99 (1); Lys 1.01 (1); NH 3 0.96 (1); hArg (Et2) 1.02 (1); Pal (3) 2.30 (2); p-F-Phe 1.15 (1); Nal (2) 0.96 (1).

39 (RS-12972): Ser 0.76 (1); Pro 1.06 (1); Ala 0.89 (1); Leu 1.04 (1); Tyr 1.07 (1); 35 p-Cl-Phe 1.17 (1); NH3 1.03 (1);

Pal (3) 2.10 (2); hArg (C 2 H 5 O 2: CH 2 CF 3) 1.66 (1); Nal (2) 1.14 (1).

II

,. '· "7 S

49 yοζο

Biological Activity of Compounds (a) Decreased Testosterone Levels Decreased testosterone levels were measured using the following test procedure: 5 Two studies were performed in one of 11 and 13 full-grown, male, intact be-agle dogs (iåltSSn 1.5-6.5 years, supplied by Syntex or White Eagle). For 6 weeks each week, the animals were randomly assigned to one of 11 or 10 13 dose groups (excipient or different doses of each LHRH antagonist) so that 6 dogs were included in each group. Group 1 patients received a single subcutaneous injection of 0.1 ml of excipient per kg of body weight. Animals in the other groups received the doses shown in List 1 below (μg compound / 0.1 ml injection volume / kg body weight). The excipient was a 1: 1 mixture of propylene glycol and physiological saline. A blood sample of approximately 3 ml of heparin was taken from each dog through the artery just prior to injection and 30 ml to 20 minutes sec 1, 2, 4, 6, 8 and 24 hours after injection into the injection. The blood sample was randomly placed in Jai after collection. Plasma was separated by centrifugation and suction filtration for 2 hours. and stored at -20 ° C prior to RIA determination of testosterone levels.

Testosterone levels were measured by radioimmunoassay. The results of the two studies were combined, and testosterone levels plotted as a function of dose levels were obtained to obtain Kayra ID ^ Q values (dose required to reduce testosterone levels to 50% of their maximum value in 8 hours). These values are shown in Table 3, which also shows the potency against the standard LHRH antagonist Detirelix (RS-68439).

50 'J' / 3 /! ^

List 1 Dose groups

Study 1 (11 dose groups):

Group Compound (RS No.) Dose 1 Element 2 RS-26306 0.06yg / 0.1ml / kg 3 RS-26306 0.25yg / 0.1ml / kg 4 RS-26306 1.0yg / 0.1l./l/ kg 5 RS-26306 4 10yg / 0, lul / kg 6 RS-26306 16.0y3 / 0.1 lml / kg 7 RS-26723 0f06yg / 0.1 lml / kg 8 RS-26723 0; 25yQ / 0, lml / kg 9 RS-26723 1f0yg / 0.1ml / kg 10 RS-26723 4, Oyg / QfIml / kg 11 RS-26723 16; Oyg / ύ.1ml / kg

Study 2 (13 dose groups)

Group Compound (RS No.) Dose 1 Element 2 RS-68439 (standard!) 0.25 ug / 0.1 ml / kg 3 RS-68439 (standard!) 1.0 ug / 0 imi / kg 4 RS-68439 ( standard!) 4.0 ug / 0.1 ml / kg: 5 RS-68439 (standard!) 16.0 ug / 0.1 ml / kg 6 RS-15378 0.25 ug / 0.1 ml / kg 7 RS-15378 1.0 μg / 0.1 lmi / kg δ RS-15378 4, γ yg / 0 lml / kg 9 RS-15378 16.0 μg / 0.1 lml / kg 10 RS-15678 ° »2S pg / 0.1l / kg .1 '11 RS-15678 1.0 Ug / 0 lml / kg 12 RS-15678 4.0 ug / 0.1 lml / kg 13 RS-15i78 ug / ojlml / kg 92326 51 C Λ α> ^ c 00 5 ι ^ ^ ^ '2 Ο Ο <3 * CNJ CO C0 Ε · * · ·> * »· *» ** Ο) Ο <-Η Ο —I ΙΑ Γ- C -Η · * - ν— - ^ ^ -— m -Ρ

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Using the following procedures, the histamine-5 release and anti-ovulatory activity of typical compounds of this invention were compared.

1. Histamine release study

Miscellaneous peritoneal cells were isolated from three or four male Sprague-Dawley rats (350 g, Iffa-Credo, Ransa) by intraperitoneal injection of 10 ml of 0.9% NaCl (containing 50 μg heparin / ml). ). After lightly rubbing the abdominal cavity for 1 minute, the peritoneal cells were isolated and pooled and centrifuged for 5 minutes at 1500 min. Three times with Ca ++ - free Krebs-Ringer-15 buffer (KRB) having the following composition: 141, 9 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO 4, 2.5 mM Na 2 HPO 4, 0.6 μM KH 2 PO 4; after rinsing, the cells were counted under a microscope and diluted with an appropriate volume of Ca-free KBR to achieve a cell density of about 2 x 10® cells / ml. A 0.3 ml aliquot was preheated with 0.3 ml of Ca ++ - free KRB for 5 minutes at 35 ° C, and 0.1 ml of the appropriate test drug solution was added. After 5 minutes, the reaction was started by adding 0.1 ml of Ca-containing KRB to reach the desired concentration. 25 sex. After 15 minutes, the reaction was quenched by the addition of 2.5 mL of ice-cold Ca ++ -free KRB and placing the suspension on them. After centrifugation of the cell suspension, the histamine content of the supernatant was determined fluorometrically by the method of Shore et al. 30 / Immunol. 127 (1959) 182-1867 »omitting the extraction process (excitation at 365 nm, emission at 450 nm). None of the reagents fluoresced with O-phthalaldialdehyde at the concentrations used in the experiments.

The total histamine content of the cell suspension was determined after sonication (2 min, pulse rate 5 / s).

Spontaneous histamine release was subtracted from all measured values.

5 - Invoices

Percent histamine release was calculated using the following equation:

Spontaneous release of histamine from cells in free supernatant 10 - x 100

Total histamine in cell suspension - Reagents and equipment: O-phthalic dialdehyde was purchased from Fluka (No. 79760). All other compounds were of analytical grade and dissolved in Ca ++ - free KRB. The fluorimeter used was a Perkin Elmer 2000 model. UltraSSn treatment was performed with a VIBRA-CELL (Sonics Materials) using a microcarrier.

20 2. Investigation of anti-ovulation

Procedure: Female rats are adapted to laboratory conditions (light to dark ratio 14:10, lights on chlorine 5 in the morning) vShintSan for 7-10 days. For each rat, perform a daily mother rinse from 7.30 to 9.00 for at least 12 days. The cytology of the rinsing fluid is examined microscopically to determine the phase of the estrus cycle. Rats that have had at least 2 normal four-day cycles before the current cycle are selected. On the morning of the expected day of estrus, the rats are sacrificed, the fallopian tubes are removed, and the presence of newly ejaculated oocytes is examined. The eggs are "attracted" out of the fallopian tube and counted. The percentage of females experiencing ovulation is plotted as a function of the logarithm of the dose to calculate an ED50 for the anti-ovulatory effect.

The histamine release effect and the anti-ovulatory effect are shown in Table 4 below.

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Acute toxicity of the compounds

The acute toxicity of the compounds of the invention was assessed by intravenous injection to rats. Groups of 3 male rats received a single injection of 5 agents or drugs (RS-15378 or RS-26306) at a dose of 1.0 mg / kg. The other two groups of rats received either 0.3 mg or 1.0 mg of standard compound (RS-68439) / kg.

(All compounds are named in the previous examples).

Injections were given into the dental vein every 10 to 20 seconds. Animals were monitored for clinical signs of toxicity at randomly after administration and at 0.5, 1.0, 3.0, and 6.0 hours after injection.

The results are shown in Table 5.

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Physiological formulas

The following are typical physiological compositions which contain as an active ingredient an LHRH antagonist according to the invention, for example 5 (N-Ac-D-Nal (2) 1, D-pCl-Phe2, Pal (3) 3 '). 6, Bth8, D-Ala10) - LHRH as such or as a physiologically acceptable salt thereof, for example, as an acetic acid addition salt, a zinc salt, a zinc tannate salt and the like.

A. Tablet Formulations 1. LHRH antagonist 10.0 mg

Compressible sugar, USP 86.0 mg

Calcium stearate 4.0 mg 2. LHRH antagonist 10.0 mg

Compressible sugar, USP 88.5 mg 15 Magnesium stearate 1.5 mg 3. LHRH antagonist 5.0 mg

Mannitol, USP 83.5 mg

Magnesium stearate, USP 1.5 mg

Pregelatinized observation, USP 10.0 mg 20 4. LHRH antagonist 10.0 mg

Lactose, USP 74.5 mg

Pregelatinized starch, USP 15.0 mg

Magnesium Stearate, USP 1.5 mg Method of Preparation (a) LHRH antagonist! dissolve in a sufficient amount of water to form a brandy granulate when mixed with a portion of the sugar excipients. After complete mixing, the granulate is dried on a plate or in a fluid bed dryer. The dry granulate is screened to break up any large aggregates and then mixed with the remaining ingredients. The granulate is then compressed into tablets having a defined mass on a conventional tableting machine.

35 (b) In this method of preparation, all formulations contain 0.01% containing 0.01% gelatin (USP). Gel 58 92376 latin is first dissolved in water as a granulation solvent, and an LHRH analog is dissolved in its fraction. The remaining steps are at the same time as in (a).

Formula 4 could be used as a mytts oral tablet.

B. Long-acting Intramuscular Injection Formula 1. Long-acting Inhalation Sesame Diesel Gel 10 LHR Antagonist 10.0 mg

Aluminum monostearate, USP 20.0 mg

SeesamiOljya gs gives 1.0 ml

The aluminum monostearate and sesame oil are combined and the combination is heated at 125 ° C with stirring until a clear yellow solution is formed. The tSma mixture is then autoclaved to perform sterilization and allowed to cool. The LHRH analog is then added aseptically while triturating. Particularly preferred LHRH analogs are sparingly soluble salts, for example, zinc-20 salts, zinc tannate salts, pamoate salts and the like. The Nile has an exceptionally long duration of action.

2. Long-acting intramuscular injection microcapsules made of biodegradable polymer. 25 LHRH antagonists 7% 25/75 glycoside / actide copolymer 93% (intrinsic viscosity 0.5)

The microcapsules of the above formula are suspended in the following solution: Dextrose 5.0%; 0.5% sodium CMC

Benzyl alcohol 0.9%

Tween 80 0.1%

Purified water to gs 100.0% 35 25 mg microcapsules were suspended in 1.0 ml of vehicle.

92326 59 C. Aqueous intramuscular injection of LHRH antagonist 0.5%

Acetic acid 0.02 mol / l

Benzylalkanol 0.9% 5 Mannitol 3.5%

Propylene glycol 20%

NaOH to bring the pH to 5

Vetta gs to 100%

Acetic acid, benzyl alcohol, mannitol and pro-10 methylene glycol were dissolved in 90% water. The LHRH antagonist was then dissolved in the paste solution, and the pH was adjusted with NaOH. The missing amount of water was added. The solution was filtered through a 1 μm filter, packaged in small vials and sterilized by autoclaving.

15 D. Nasal aqueous formula LHRH antagonist 50 mg 0.02M acetate buffer 5 ml

Sodium glycocholate 500 mg 0.02M acetate buffer, pH 5.2 to 10 ml 20 E. Formula for rectal administration

Suppository: LHRH antagonist 5.0 mg

Witepsol H15 20.0 g of LHRH antagonist was combined with molten Witepsol .25 H15, mixed well and poured into 2 g molds.

·

Claims (10)

60 92326 A nonapeptide or decapeptide analogue of LHRH of formula (I), ABC-Ser-DEFG-Pro-J (I) 123 4 5678 9 10 or a physiologically acceptable salt thereof, expressed as net -10 that A is one of the aminoacyl groups N-Ac-3- (1-naphthyl) -D, L-alanyl and N-Ac-3- (2-naphthyl) -D, L-alanyl, their D- or L- isomer; B is one of the aminoacyl groups D-phenylalanyl, D-p-chlorophenylalanyl, D-p-fluorophenylalanyl and D-α-methyl-p-chlorophenylalanyl; C is one of the aminoacyl groups D-tryptophanyl and 3- (3-pyridyl) -D-alanyl; D is one of the aminoacyl groups L-phenylalanyl, L-tyrosyl, 3- (3-pyridyl) alanyl and arginyl, or an amino-20-acyl group having the structural formula -HN-CH-CO- (™ 2'n UI) NH 1 1 2 R is -HN-C = NR wherein n is 1 to 5; R 1 is alkyl containing 1 to 6 carbon atoms, or fluoroalkyl; and R 2 is hydrogen or R 1; E is 3- (3-pyridyl) -D-alanyl, D-tyrosyl, D-trypto-α-30yl or an aminoacyl group having the structural formula -HN-CH-CO- (CB2) n (ID NH 1 1 2 35 R -HN-C = NR 61 C o 7 O ^ ^ o where n is 1 to 5; R 1 is alkyl containing 1 to 6 carbon atoms, and R 2 is hydrogen or R 1; where R1-HN-C = NR2 is 2. A is N-Ac-D-Nal (2); B is D-pCl-Phe; C is D-Trp or D-Pal (3); D is Tyr, Arg, Deh, Mbh or Bth; F is Leu; and J is D-AlaNH2. Compound according to Claim 2, characterized in that it is N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Pal (3) - Leu-Pro-D-AlaNH2 or a physiologically acceptable salt thereof. A compound according to claim 1 or a physiologically acceptable salt thereof, characterized in that Compound according to Claim 2, characterized in that it is N-Ac-D-Nal (2) -D-pCl-Phe-D-62 9 'i 3' ί 6 Pal (3) -Ser-Tyr- D-Pal (3) -Leu-Deh-Pro-D-AlaNH2 or a physiologically acceptable salt thereof. 5 Pal (3) -Ser-Mbh-D-Pal (3) -Leu-Mbh-Pro-D-AlaNH2 or a physiologically acceptable salt thereof. A compound according to claim 2, characterized in that it is N-Ac-D-Nal (2) -D-pCl-Phe-D- 5 HN N \ / H where m is 1 to 4; 10. is one of the aminoacyl groups L-leucyl and L-phenylalanyl; G is an aminoacyl group of structural formula -HN-CH-CO- (CH2) n (II) NH 1 * 2 R -HN-C = NR wherein n is 1 to 5; R 1 is alkyl containing 1 to 6 carbon atoms, or fluoroalkyl; and R 2 is hydrogen or R 1; and J is D-alaninamide or glycinamide. A compound according to claim 2, characterized in that it is N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Pal (3) -D-Pal (3) -Leu-Bth-Pro-D-AlaNH2 or a physiologically acceptable salt thereof. Compound according to Claim 2, characterized in that it is N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Arg-D-Pal (3) -Leu-Deh-Pro-D-AlaNH2 or a physiologically acceptable salt thereof. Compound according to Claim 2, characterized in that it is N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Bth-D-Tyr-Leu-Bth -Pro-D-AlaNH2 or a physiologically acceptable salt thereof. A compound according to claim 2, characterized in that it is N-Ac-D-Nal (2) -D-pCl-Phe-D-Pal (3) -Ser-Tyr-D-Deh-Leu -Deh-Pro-D-AlaNH2 or a physiologically acceptable salt thereof. Composition, characterized in that it contains a compound according to one of Claims 1 to 9, mixed with at least one physiologically acceptable excipient. 63 92326